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When a high-fidelity DNA polymerase encounters certain DNA-damage sites, its progress can be stalled and one or more lesion-bypass polymerases are recruited to transit the lesion. Here, we consider two representative types of lesions: (i) 7,8-dihydro-8-oxoguanine (8-oxoG), a small, highly prevalent lesion caused by oxidative damage; and (ii) bulky lesions derived from the environmental pre-carcinogen benzo[a]pyrene, in the high-fidelity DNA polymerase Bacillus fragment (BF) from Bacillus stearothermophilus and in the lesion-bypass DNA polymerase IV (Dpo4) from Sulfolobus solfataricus. The tight fit of the BF polymerase around the nascent base pair contrasts with the more spacious, solvent-exposed active site of Dpo4, and these differences in architecture result in distinctions in their respective functions: one-step versus stepwise polymerase translocation, mutagenic versus accurate bypass of 8-oxoG, and polymerase stalling versus mutagenic bypass at bulky benzo[a]pyrene-derived lesions.

Equilin and equilenin, components of the hormone replacement therapy drug Premarin, can be metabolized to the catechol 4-hydroxyequilenin (4-OHEN). The quinoids produced by 4-OHEN oxidation react with dC, dA, and dG to form unusual stable cyclic adducts, which have been found in human breast tumor tissue. Four stereoisomeric adducts have been identified for each base. These 12 Premarin-derived adducts provide a unique opportunity for analyzing effects of stereochemistry and base damage on DNA structure and consequently its function. Our computational studies have shown that these adducts, with obstructed Watson-Crick hydrogen-bond edges and near-perpendicular ring systems, have limited conformational flexibility and near-mirror-image conformations in stereoisomer pairs. The dC and dA adducts can adopt major- and minor-groove positions in the double helix, but the dG adducts are positioned only in the major groove. In all cases, opposite orientations of the equilenin rings with respect to the 5' --> 3' direction of the damaged strand are found in stereoisomer pairs derived from the same base, and no Watson-Crick pairing is possible. However, detailed structural properties in DNA duplexes are distinct for each stereoisomer of each damaged base. These differences may underlie observed differential stereoisomer and base-dependent mutagenicities and repair susceptibilities of these adducts.

The Y-family DNA polymerase Dpo4, from the archaeon bacterium Sulfolobus solfataricus, is a member of the DinB family, which also contains human Pol kappa. It has a spacious active site that can accommodate two templating bases simultaneously, with one of them skipped by the incoming dNTP. Assays of single dNTP insertion opposite a benzo[ a]pyrene-derived N (2)-dG adduct, 10 S(+)- trans- anti-[BP]- N (2)-dG ([BP]G*), reveal that an incoming dATP is significantly preferred over the other three dNTPs in the TG 1*G 2 sequence context. Molecular modeling and dynamics simulations were carried out to interpret this experimental observation on a molecular level. Modeling studies suggest that the significant preference for dATP insertion observed experimentally can result from two possible dATP incorporation modes. The dATP can be inserted opposite the T on the 5' side of the adduct G 1*, using an unusual 5'-slippage pattern, in which the unadducted G 2, rather than G 1*, is skipped, to produce a -1 deletion. In addition, the dATP can be misincorporated opposite the adduct. The 5'-slippage pattern may be generally facilitated in cases where the base 3' to the lesion is the same as the adducted base.

Oxidatively generated DNA damage induced by the aromatic radical cation of the pyrene derivative 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (BPT), and by carbonate radicals anions, was monitored from the initial one-electron transfer, or hole injection step, to the formation of hot alkali-labile chemical end-products monitored by gel electrophoresis. The fractions of BPT molecules bound to double-stranded 20-35-mer oligonucleotides with noncontiguous guanines G and grouped as contiguous GG and GGG sequences were determined by a fluorescence quenching method. Utilizing intense nanosecond 355 nm Nd:YAG laser pulses, the DNA-bound BPT molecules were photoionized to BPT*+ radicals by a consecutive two-photon ionization mechanism. The BPT*+ radicals thus generated within the duplexes selectively oxidize guanine by intraduplex electron-transfer reactions, and the rate constants of these reactions follow the trend 5'-..GGG.. > 5'-..GG.. > 5'-..G... In the case of CO3*- radicals, the oxidation of guanine occurs by intermolecular collision pathways, and the bimolecular rate constants are independent of base sequence context. However, the distributions of the end-products generated by CO3*- radicals, as well as by BPT*+, are base sequence context-dependent and are greater than those in isolated guanines at the 5'-G in 5'-...GG... sequences, and the first two 5'- guanines in the 5'-..GGG sequences. These results help to clarify the conditions that lead to a similar or different base sequence dependence of the initial hole injection step and the final distribution of oxidized, alkali-labile guanine products. In the case of the intermolecular one-electron oxidant CO3*-, the rate constant of hole injection is similar for contiguous and isolated guanines, but the subsequent equilibration of holes by hopping favors trapping and product formation at contiguous guanines, and the sequence dependence of these two phenomena are not correlated. In contrast, in the case of the DNA-bound oxidant BPT*+, the hole injection rate constants, as well as hole equilibration, exhibit a similar dependence on base sequence context, and are thus correlated to one another.

The carbonate radical anion is a biologically important one-electron oxidant that can directly abstract an electron from guanine, the most easily oxidizable DNA base. Oxidation of the 5'-d(CCTACGCTACC) sequence by photochemically generated CO3*- radicals in low steady-state concentrations relevant to biological processes results in the formation of spiroiminodihydantoin diastereomers and a previously unknown lesion. The latter was excised from the oxidized oligonucleotides by enzymatic digestion with nuclease P1 and alkaline phosphatase and identified by LC-MS/MS as an unusual intrastrand cross-link between guanine and thymine. In order to further characterize the structure of this lesion, 5'-d(GpCpT) was exposed to CO3*- radicals, and the cyclic nature of the 5'-d(G*pCpT*) cross-link in which the guanine C8-atom is bound to the thymine N3-atom was confirmed by LC-MS/MS, 1D and 2D NMR studies. The effect of bridging C bases on the cross-link formation was studied in the series of 5'-d(GpC(n)pT) and 5'-d(TpC(n)pG) sequences with n = 0, 1, 2 and 3. Formation of the G*-T* cross-links is most efficient in the case of 5'-d(GpCpT). Cross-link formation (n = 0) was also observed in double-stranded DNA molecules derived from the self-complementary 5'-d(TTACGTACGTAA) sequence following exposure to CO3*- radicals and enzymatic excision of the 5'-d(G*pT*) product.

Peroxyl radicals were derived from the one-electron oxidation of polyunsaturated fatty acids by sulfate radicals that were generated by the photodissociation of peroxodisulfate anions in air-equilibrated aqueous solutions. Reactions of these peroxyl and neutral guanine radicals, also generated by oxidation with sulfate radicals, were investigated by laser kinetic spectroscopy, and the guanine oxidation products were identified by HPLC and mass spectrometry methods. Sulfate radicals rapidly oxidize arachidonic (ArAc), linoleic (LnAc), and palmitoleic (PmAc) acids with similar rate constants, (2-4) x 10 (9) M (-1) s (-1). The C-centered radicals derived from the oxidation of ArAc and LnAc include nonconjugated Rn(.) ( approximately 80%) and conjugated bis-allylic Rba(.) ( approximately 20%) radicals. The latter were detectable in the absence of oxygen by their prominent, narrow absorption band at 280 nm. The Rn(.) radicals of ArAc (containing three bis-allylic sites) transform to the Rba(.) radicals via an intramolecular H-atom abstraction [rate constant (7.5 +/- 0.7) x 10 (4) s (-1)]. In contrast, the Rn(.) radicals of LnAc that contain only one bis-allylic site do not transform intramolecularly to the Rba(.) radicals. In the case of PmAc, which contains only one double bond, the Rba(.) radicals are not observed. The Rn(.) radicals of PmAc rapidly combine with oxygen with a rate constant of (3.8 +/- 0.4) x 10(9) M(-1) s(-1). The Rba(.) radicals of ArAc are less reactive and react with oxygen with a rate constant of (2.2 +/- 0.2) x 10 (8) M (-1) s (-1). The ArAc peroxyl radicals formed spontaneously eliminate superoxide radical anions [rate constant = (3.4 +/- 0.3) x 10 (4) M (-1) s (-1)]. The stable oxidative lesions derived from the 2',3',5'-tri- O-acetylguanosine or 2',3',5'-tri- O-acetyl-8-oxo-7,8-dihydroguanosine radicals and their subsequent reactions with ArAc peroxyl radicals were also investigated. The major products found were the 2,5-diamino-4 H-imidazolone (Iz), dehydroguanidinohydantoin (Gh ox), and diastereomeric spiroiminodihydantoin (Sp) nucleosides from 2',3',5'-tri- O-acetylguanosine and the Gh ox and Sp nucleosides from 2',3',5'-tri- O-acetyl-8-oxo-7,8-dihydroguanosine. In air-saturated aqueous solutions, covalent alkylated guanine adducts were not detected.

It has now been nearly two decades since the first solution structures of DNA duplexes covalently damaged by metabolically activated polycyclic aromatic hydrocarbons and amines were determined by NMR. Dozens of such high-resolution structures are now available, and some broad structural themes have been uncovered. It has been hypothesized that the solution structures are relevant to the biochemical processing of the adducts. The structural features of the adducts are considered to determine their mutational properties in DNA polymerases and their repair susceptibilities. In recent years, a number of crystal structures of DNA adducts of interest to our work have been determined in DNA polymerases. Accordingly, it is now timely to consider how NMR solution structures relate to structures within DNA polymerases. The NMR solution structural themes for polycyclic aromatic adducts are often observed in polymerase crystal structures. While the polymerase interactions can on occasion override the solution preferences, intrinsic adduct conformations favored in solution are often manifested within polymerases and likely play a significant role in lesion processing.

The nucleotide excision repair (NER) machinery excises a variety of bulky DNA lesions, but with varying efficiencies. The structural features of the DNA lesions that govern these differences are not well understood. An intriguing model system for studying structure-function relationships in NER is the major adduct derived from the reaction of the highly tumorigenic metabolite of benzo[a]pyrene, (+)-anti-benzo[a]pyrene diol epoxide, with the exocyclic amino group of guanine ((+)-trans-anti-[BP]-N(2)-dG, or G*). The rates of incision of the stereochemically identical lesions catalyzed by the prokaryotic UvrABC system was shown to be greater by a factor of 2.3+/-0.3 in the TG*T than in the CG*C sequence context [Biochemistry 46 (2007) 7006-7015]. Here we employ molecular dynamics simulations to elucidate the origin of the greater excision efficiency in the TG*T case and, more broadly, to delineate structural parameters that enhance NER. Our results show that the BP aromatic ring system is 5'-directed along the modified strand in the B-DNA minor groove in both sequence contexts. However, the TG*T modified duplex is much more dynamically flexible, featuring more perturbed and mobile Watson-Crick hydrogen bonding adjacent to the lesion, a greater impairment in stacking interactions, more dynamic local roll/bending, and more minor groove flexibility. These characteristics explain a number of experimental observations concerning the (+)-trans-anti-[BP]-N(2)-dG adduct in double-stranded DNA with the TG*T sequence context: its conformational heterogeneity in NMR solution studies, its highly flexible bend, and its lower thermal stability. By contrast, the CG*C modified duplex is characterized by a single BP conformation and a rigid bend. While current recognition models of bulky lesions by NER factors have stressed the importance of impaired Watson-Crick pairing/stacking and bending, our results highlight the likelihood of an important role for the local dynamics in the vicinity of the lesion.

Oxidatively generated damage to DNA induced by a pyrenyl photosensitizer residue (Py) covalently attached to a guanine base in the DNA sequence context 5'-d(CAT[G1Py]CG2TCCTAC) in aerated solutions was monitored from the initial one-electron transfer, or hole injection step, to the formation of chemical end-products monitored by HPLC, mass spectrometry, and high-resolution gel electrophoresis. Hole injection into the DNA was initiated by two-photon excitation of the Py residue with 355 nm laser pulses, thus producing the radical cation Py*+ and hydrated electrons; the latter are trapped by O2, thus forming the superoxide anion O2*-. The decay of the Py*+ radical is correlated with the appearance of the G*+/G(-H)* radical on microsecond time scales, and O2*- combines with guanine radicals at G1 to form alkali-labile 2,5-diamino-4H-imidazolone lesions (Iz1Py). Product formation in the modified strand is smaller by a factor of 2.4 in double-stranded than in single-stranded DNA. In double-stranded DNA, hot piperidine-mediated cleavage at G2 occurs only after G1Py, an efficient hole trap, is oxidized thus generating tandem lesions. An upper limit of hole hopping rates, khh < 5 x 103 s-1 from G1*+-Py to G2 can be estimated from the known rates of the combination reaction of the G(-H)* and O2*- radicals. The formation of Iz products in the unmodified complementary strand compared to the modified strand in the duplex is approximately 10 times smaller. The formation of tandem lesions is observed even at low levels of irradiation corresponding to "single-hit" conditions when less than approximately 10% of the oligonucleotide strands are damaged. A plausible mechanism for this observation is discussed.

Benzo[a]pyrene (B[a]P), a known environmental pollutant and tobacco smoke carcinogen, is metabolically activated to highly tumorigenic B[a]P diol epoxide derivatives that predominantly form N(2)-guanine adducts in cellular DNA. Although nucleotide excision repair (NER) is an important cellular defense mechanism, the molecular basis of recognition of these bulky lesions is poorly understood. In order to investigate the effects of DNA adduct structure on NER, three stereoisomeric and conformationally different B[a]P-N(2)-dG lesions were site specifically incorporated into identical 135-mer duplexes and their response to purified NER factors was investigated. Using a permanganate footprinting assay, the NER lesion recognition factor XPC/HR23B exhibits, in each case, remarkably different patterns of helix opening that is also markedly distinct in the case of an intra-strand crosslinked cisplatin adduct. The different extents of helix distortions, as well as differences in the overall binding of XPC/HR23B to double-stranded DNA containing either of the three stereoisomeric B[a]P-N(2)-dG lesions, are correlated with dual incisions catalyzed by a reconstituted incision system of six purified NER factors, and by the full NER apparatus in cell-free nuclear extracts.

The influence of DNA base sequence context on the removal of a bulky benzo[a]pyrene diol epoxide-guanine adduct, (+)-trans-B[a]P-N2-dG (G*), by UvrABC nuclease from the thermophilic organism Bacillus caldotenax was investigated. The lesion was flanked by either T or C in otherwise identical complementary 43-mer duplexes (TG*T or CG*C, respectively). It was reported earlier that in the CG*C context, a dominant minor groove adduct structure was observed by NMR methods with all Watson-Crick base pairs intact, and the duplex exhibited a rigid bend. In contrast, in the TG*T context, a highly flexible bend was observed, base pairing at G*, and two 5'-base pairs flanking the adduct were impaired, and multiple solvent-accessible adduct conformations were observed. The TG*T-43-mer duplexes are incised with consistently greater efficiency by UvrABC proteins from B. caldotenax by a factor of 2.3 +/- 0.3. The rates of incisions increase with increasing temperature and are characterized by linear Arrhenius plots with activation energies of 27.0 +/- 1.5 and 23.4 +/- 1.0 kcal/mol for CG*C and TG*T duplexes, respectively. These values reflect the thermophilic characteristics of the UVrABC nuclease complex and the contributions of the different DNA substrates to the overall activation energies. These effects are consistent with base sequence context-dependent differences in structural disorder engendered by a loss of local base stacking interactions and Watson-Crick base pairing in the immediate vicinity of the lesions in the TG*T duplexes. The local weakening of base pairing interactions constitutes a recognition element of the UvrABC nucleotide excision repair apparatus.

Base excision repair (BER) is the major pathway employed to excise oxidized DNA lesions. Human Neil1, a versatile glycosylase in the BER pathway, repairs a diverse array of oxidative lesions; however, the most prevalent, 8-oxo-7,8-dihydroguanine (8-oxoG), is only weakly excised. The structural origin of hNeil1's ability to repair a variety of lesions but not 8-oxoG is a model system for connecting enzyme structure and lesion-recognition specificity. To elucidate structural properties determining hNeil1's substrate specificities, we have investigated it in complex with two pairs of representative well-repaired substrates: the R- and S-spiroiminodihydantoin (Sp) stereoisomers, nonplanar further oxidation products of guanine, and the 5R,6S- and 5S,6R-thymine glycol (Tg) stereoisomers, the most prevalent oxidative lesions of thymine. We also investigate the poorly repaired 8-oxoG. We employed molecular modeling and 10 ns molecular dynamics (MD) simulations. The results of our investigations provide structural explanations for the ability of hNeil1 to excise a variety of oxidative lesions: they possess common chemical features, namely, a pyrimidine-like ring and shared hydrogen bond donor-acceptor properties, which allow the lesions to fit well in the binding pocket, which is somewhat flexible. However, the planar 8-oxoG is not as well accommodated in the shallow and comparatively cramped recognition pocket; it has fewer hydrogen bonding interactions with the enzyme and a solvent exposed six-membered ring, consistent with its poor repair susceptibility by this enzyme.

The biologically most significant genotoxic metabolite of the environmental pollutant benzo[a]pyrene (B[a]P), (+)-7R,8S-diol 9S,10R-epoxide, reacts chemically with guanine in DNA, resulting in the predominant formation of (+)-trans-B[a]P-N(2)-dG and, to a lesser extent, (+)-cis-B[a]P-N(2)-dG adducts. Here, we compare the effects of the adduct stereochemistry and conformation on the methylation of cytosine catalyzed by two purified prokaryotic DNA methyltransferases (MTases), SssI and HhaI, with the lesions positioned within or adjacent to their CG and GCGC recognition sites, respectively. The fluorescence properties of the pyrenyl residues of the (+)-cis-B[a]P-N(2)-dG and (+)-trans-B[a]P-N(2)-dG adducts in complexes with MTases are enhanced, but to different extents, indicating that aromatic B[a]P residues are positioned in different microenvironments in the DNA-protein complexes. We have previously shown that the (+)-trans-isomeric adduct inhibits both the binding and methylating efficiencies (k(cat)) of both MTases [Subach OM, Baskunov VB, Darii MV, Maltseva DV, Alexandrov DA, Kirsanova OV, Kolbanovskiy A, Kolbanovskiy M, Johnson F, Bonala R, et al. (2006) Biochemistry45, 6142-6159]. Here we show that the stereoisomeric (+)-cis-B[a]P-N(2)-dG lesion has only a minimal effect on the binding of these MTases and on k(cat). The minor-groove (+)-trans adduct interferes with the formation of the normal DNA minor-groove contacts with the catalytic loop of the MTases. However, the intercalated base-displaced (+)-cis adduct does not interfere with the minor-groove DNA-catalytic loop contacts, allowing near-normal binding of the MTases and undiminished k(cat) values.

The Y-family DNA polymerase Dpo4, from the thermophilic crenarchaeon Sulfolobus solfataricus P2, offers a valuable opportunity to investigate the effect of conformational flexibility on the bypass of bulky lesions because of its ability to function efficiently at a wide range of temperatures. Combined molecular modeling and experimental kinetic studies have been carried out for 10S-(+)-trans-anti-[BP]-N2-dG ((+)-ta-[BP]G), a lesion derived from the covalent reaction of a benzo[a]pyrene metabolite with guanine in DNA, at 55 degrees C and results compared with an earlier study at 37 degrees C (Perlow-Poehnelt, R. A., Likhterov, I., Scicchitano, D. A., Geacintov, N. E., and Broyde, S. (2004) J. Biol. Chem. 279, 36951-36961). The experimental results show that there is more overall nucleotide insertion opposite (+)-ta-[BP]G due to particularly enhanced mismatch incorporation at 55 degrees C compared with 37 degrees C. The molecular dynamics simulations suggest that mismatched nucleotide insertion opposite (+)-ta-[BP]G is increased at 55 degrees C compared with 37 degrees C because the higher temperature shifts the preference of the damaged base from the anti to the syn conformation, with the carcinogen on the more open major groove side. The mismatched dNTP structures are less distorted when the damaged base is syn than when it is anti, at the higher temperature. However, with the normal partner dCTP, the anti conformation with close to Watson-Crick alignment remains more favorable. The molecular dynamics simulations are consistent with the kcat values for nucleotide incorporation opposite the lesion studied, providing structural interpretation of the experimental observations. The observed temperature effect suggests that conformational flexibility plays a role in nucleotide incorporation and bypass fidelity opposite (+)-ta-[BP]G by Dpo4.

The equine estrogens, equilin and equilenin, are major components of the drug Premarin, the most widely used formula for hormone replacement therapy. The derivative 4-hydroxyequilenin (4-OHEN), a major phase I metabolite of equilin and equilenin, autoxidizes to potent cytotoxic quinoids that can react in vitro and in vivo with cytosine and adenine in DNA. Unique cyclic adducts containing the same bicyclo[3.3.1]nonane-type connection ring are produced. Each base adduct has four stereoisomers. In order to elucidate the structural effects of A versus C modification, we have carried out molecular dynamics simulations of the stereoisomeric 4-OHEN-A adducts in DNA 11-mer duplexes and compared results with an earlier study of the C adducts (Ding, S., Shapiro, R., Geacintov, N.E., and Broyde, S. (2005) Equilenin-Derived DNA Adducts to Cytosine in DNA Duplexes: Structures and Thermodynamics, Biochemistry 44, 14565-14576). Similar stereochemical principles govern the orientations in DNA duplexes of the 4-OHEN-A adducts as for the analogous C adducts, with opposite orientations of the equilenin rings in stereoisomeric pairs of adducts characterized by near-mirror image circular dichroism (CD) spectra. However, the larger purine adducts have unique structural properties in the duplexes that distinguish their characteristics from those of the pyrimidine adducts. Significant differences are observed in terms of hydrogen bonding, stacking, bending, groove dimensions, solvent exposure, and hydrophobic interactions; also, each of the four stereoisomeric 4-OHEN-A adducts exhibit distinct structural features. Each base adduct and stereoisomer distorts the structure of the DNA duplex differently. These characteristics may manifest themselves in terms of differential nucleotide excision repair susceptibilities and mutagenic activities of the 4-OHEN-A and C adducts.

The environmental carcinogen benzo[a]pyrene (BP) is metabolized to reactive diol epoxides that bind to cellular DNA by predominantly forming N2-guanine adducts (G*). Mutation hotspots for these adducts are frequently found in 5'- ... GG ... dinucleotide sequences, but their origins are poorly understood. Here we used high resolution NMR and molecular dynamics simulations to investigate differences in G* adduct conformations in 5'- ... CG*GC ... and 5'- ... CGG* C... sequence contexts in otherwise identical 12-mer duplexes. The BP rings are positioned 5' along the modified strand in the minor groove in both cases. However, subtle orientational differences cause strong distinctions in structural distortions of the DNA duplexes, because the exocyclic amino groups of flanking guanines on both strands compete for space with the BP rings in the minor groove, acting as guideposts for placement of the BP. In the 5'- ... CGG* C ... case, the 5'-flanking G . C base pair is severely untwisted, concomitant with a bend deduced from electrophoretic mobility. In the 5'- ... CG*GC ... context, there is no untwisting, but there is significant destabilization of the 5'-flanking Watson-Crick base pair. The minor groove width opens near the lesion in both cases, but more for 5'- ... CGG*C.... Differential sequence-dependent removal rates of this lesion result and may contribute to the mutation hotspot phenomenon.

Peroxynitrite is produced during inflammation and combines rapidly with carbon dioxide to yield the unstable nitrosoperoxycarbonate, which decomposes (in part) to CO(3) (.-) and (.)NO(2) radicals. The CO(3) (.-) radicals oxidize guanine bases in DNA through a one-electron transfer reaction process that ultimately results in the formation of stable guanine oxidation products. Here we have explored these mechanisms, starting with a spectroscopic study of the kinetics of electron transfer from 20-22mer double-stranded oligonucleotides to CO(3) (.-) radicals, together with the effects of base sequence on the formation of the end-products in runs of one, two, or three contiguous guanines. The distributions of these alkali-labile lesions were determined by gel electrophoresis methods. The cascade of events was initiated through the use of 308 nm XeCl excimer laser pulses to generate CO(3) (.-) radicals by an established method based on the photodissociation of persulfate to sulfate radicals and the oxidation of bicarbonate. Although the Saito model (Saito et al., J. Am. Chem. Soc. 1995, 117, 6406-6407) predicts relative ease of one-electron oxidations in DNA, following the trend 5'-GGG > 5'-GG > 5'-G, we found that the rate constants for CO(3) (.-)-mediated oxidation of guanines in these sequence contexts (k(5)) showed only small variation within a narrow range [(1.5-3.0)x10(7) M(-1) s(-1)]. In contrast, the distributions of the end-products are dependent on the base sequence context and are higher at the 5'-G in 5'-GG sequences and at the first two 5'-guanines in the 5'-GGG sequences. These effects are attributed to a combination of initial hole distributions among the contiguous guanines and the subsequent differences in chemical reaction yields at each guanine. The lack of dependence of k(5) on sequence context indicates that the one-electron oxidation of guanine in DNA by CO(3) (.-) radicals occurs by an inner-sphere mechanism.

We have investigated how a benzo[a]pyrene-derived N2-dG adduct, 10S(+)-trans-anti-[BP]-N2-dG ([BP]G*), is processed in a well-characterized Pol I family model replicative DNA polymerase, Bacillus fragment (BF). Experimental results are presented that reveal relatively facile nucleotide incorporation opposite the lesion, but very inefficient further extension. Computational studies follow the possible bypass of [BP]G* through the pre-insertion, insertion and post-insertion sites as BF alternates between open and closed conformations. With dG* in the normal B-DNA anti conformation, BP seriously disturbs the polymerase structure, positioning itself either deeply in the pre-insertion site or on the crowded evolving minor groove side of the modified template, consistent with a polymerase-blocking conformation. With dG* in the less prevalent syn conformation, BP causes less distortion: it is either out of the pre-insertion site or in the major groove open pocket of the polymerase. Thus, the syn conformation can account for the observed relatively easy incorporation of nucleotides, with mutagenic purines favored, opposite the [BP]G* adduct. However, with the lesion in the BF post-insertion site, more serious distortions caused by the adduct even in the syn conformation explain the very inefficient extension observed experimentally. In vivo, a switch to a potentially error-prone bypass polymerase likely dominates translesion bypass.

Guanine in DNA is a major oxidation target owing to its low ionization potential (IP), and there is often an inverse correlation between damage frequency and sequence-dependent variation in guanine IP. We report that the biological oxidant nitrosoperoxycarbonate (ONOOCO2(-)) paradoxically selects guanines with the highest IP in GC-containing contexts. Along with sequence-dependent variation in damage chemistry, this behavior points to factors other than charge migration as determinants of genomic DNA oxidation.

The diastereomeric spiroiminodihydantoin (Sp) lesions are oxidation products of guanine and 8-oxo-7,8-dihydroguanine (8-oxoG) and have generated considerable interest because of their stereochemistry-dependent mutagenic properties in vivo (Henderson, P. T., et al. (2003) Biochemistry 42, 9257-9262). However, the absolute configurations of the two diastereomers have not yet been elucidated, and such information may prove valuable for understanding relationships between biological function and structure at the DNA level (Jia, L., Shafirovich, V., Shapiro, R., Geacintov, N. E., and Broyde, S. (2005) Biochemistry 44, 13342-13353). We have synthesized the two chiral Sp nucleobases by hydrolysis of the nucleosides denoted by dSp1 and dSp2 according to their elution order in HPLC experiments using a Hypercarb column, and determined their absolute configurations using a combination of experimentally measured optical rotatory dispersion (ORD) spectra in aqueous solutions and computed ORD specific rotations using density functional theory (DFT). Recent developments have shown that DFT methods are now sufficiently robust for predicting ORD values of chiral molecules (Polavarapu, P. L. (2002) Chirality 14, 768-781). The nucleobases Sp1 and Sp2 exhibit experimentally measured CD and ORD spectra that are very close to those of the respective precursor nucleosides dSp1 and dSp2 in shape and sign. The first nucleoside stereoisomer (dSp1) to elute from a typical Hypercarb HPLC column has (-)-S, while the second (dSp2) has (+)-R absolute configuration. The R and S assignments are applicable to the amino tautomeric forms in each case.

5-Guanidino-4-nitroimidazole (NI), derived from guanine oxidation by reactive oxygen and nitrogen species, contains an unusual flexible ring-opened structure, with nitro and guanidino groups which possess multiple hydrogen bonding capabilities. In vitro primer extension experiments with bacterial and mammalian polymerases show that NI incorporates C as well as A and G opposite the lesion, depending on the polymerase. To elucidate structural and thermodynamic properties of the mutagenic NI lesion, we have investigated the structure of the modified base itself and the NI-containing nucleoside with high-level quantum mechanical calculations and have employed molecular modeling and molecular dynamics simulations in solution for the lesion in B-DNA duplexes, with four partner bases opposite the NI. Our results show that NI adopts a planar structure at the damaged base level. However, in the nucleoside and in DNA duplexes, steric hindrance between the guanidino group and its linked sugar causes NI to be nonplanar. The NI lesion can adopt both syn and anti conformations on the DNA duplex level, with the guanidino group positioned in the DNA major and minor grooves, respectively; the specific preference depends on the partner base. On the basis of hydrogen bonding and stacking interactions, groove dimensions, and bending, we find that the least distorted NI-modified duplex contains partner C, consistent with observed incorporation of C opposite NI. However, hydrogen bonding interactions between NI and partner G or A are also found, which would be compatible with the observed mismatches.

DNA damage caused by the binding of the tumorigen 7R,8S-diol 9S,10R-epoxide (B[a]PDE), a metabolite of bezo[a]pyrene, to guanine in CpG dinucleotide sequences could affect DNA methylation and, thus, represent a potential epigenetic mechanism of chemical carcinogenesis. In this work, we investigated the impact of stereoisomeric (+)- and (-)-trans-anti-B[a]P-N(2)-dG adducts (B(+) and B(-)) on DNA methylation by prokaryotic DNA methyltransferases M.SssI and M.HhaI. These two methyltransferases recognize CpG and GCGC sequences, respectively, and transfer a methyl group to the C5 atom of cytosine (C). A series of 18-mer unmethylated or hemimethylated oligodeoxynucleotide duplexes containing trans-anti-B[a]P-N(2)-dG adducts was generated. The B(+) or B(-) residues were introduced either 5' or 3' adjacent or opposite to the target 2'-deoxycytidines. The B[a]PDE lesions practically produced no effect on M.SssI binding to DNA but reduced M.HhaI binding by 1-2 orders of magnitude. In most cases, the benzo[a]pyrenyl residues decreased the methylation efficiency of hemimethylated and unmethylated DNA by M.SssI and M.HhaI. An absence of the methylation of hemimethylated duplexes was observed when either the (+)- or the (-)-trans-anti-B[a]P-N(2)-dG adduct was positioned 5' to the target dC. The effects observed may be related to the minor groove conformation of the bulky benzo[a]pyrenyl residue and to a perturbation of the normal contacts of the methyltransferase catalytic loop with the B[a]PDE-modified DNA. Our results indicate that a trans-anti-B[a]P-N(2)-dG lesion flanking a target dC in the CpG dinucleotide sequence on its 5'-side has a greater adverse impact on methylation than the same lesion when it is 3' adjacent or opposite to the target dC.

A new, highly sensitive fluorescent sensor for Zn(II) ion (a tris(2-pyridylmethyl)amine derivative) shows very strong binding and Zn(II) concentration-dependent biexponential time-resolved fluorescence (TRF) decay profiles that can be used for ratiometric estimates of Zn(II) concentrations. The ligand-metal complexes were characterized in solution by spectroscopic techniques and in the solid state by X-ray crystallography. The TRF studies revealed that the sensor aggregates in the absence of Zn(II) in a ligand concentration-dependent manner, a complication that is discerned by TRF but not by steady-state fluorescence ratiometric sensing techniques. It is shown that the same TRF methods are highly useful for monitoring Zn(II) concentrations in A549 epithelial lung cells in vitro and that the results were consistent with those in solution.

Benzo[a]pyrene is an important environmental mutagen and carcinogen. Its metabolism in cells yields the mutagenic, key ultimate carcinogen 7R,8S,9S,10R-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, (+)-anti-BPDE, which reacts via its 10-position with N2-dG in DNA to form the adduct (+)-trans-anti-BPDE-N2-dG. To gain molecular insights into BPDE-induced mutagenesis, we examined in vivo translesion synthesis and mutagenesis in yeast cells of a site-specific 10S (+)-trans-anti-BPDE-N(2)-dG adduct and the stereoisomeric 10R (-)-trans-anti-BPDE-N2-dG adduct. In wild-type cells, bypass products consisted of 76% C, 14% A and 7% G insertions opposite (+)-trans-anti-BPDE-N2-dG; and 89% C, 4% A and 4% G insertions opposite (-)-trans-anti-BPDE-N2-dG. Translesion synthesis was reduced by approximately 26-37% in rad30 mutant cells lacking Poleta, but more deficient in rev1 and almost totally deficient in rev3 (lacking Polzeta) mutants. C insertion opposite the lesion was reduced by approximately 24-33% in rad30 mutant cells, further reduced in rev1 mutant, and mostly disappeared in the rev3 mutant strain. The insertion of A was largely abolished in cells lacking either Poleta, Polzeta or Rev1. The insertion of G was not detected in either rev1 or rev3 mutant cells. The rad30 rev3 double mutant exhibited a similar phenotype as the single rev3 mutant with respect to translesion synthesis and mutagenesis. These results show that while the Polzeta pathway is generally required for translesion synthesis and mutagenesis of the (+)- and (-)-trans-anti-BPDE-N2-dG DNA adducts, Poleta, Polzeta and Rev1 together are required for G-->T transversion mutations, a major type of mutagenesis induced by these lesions. Based on biochemical and genetic results, we present mechanistic models of translesion synthesis of these two DNA adducts, involving both the one-polymerase one-step and two-polymerase two-step models.

7,8-dihydro-8-oxoguanine (oxoG), the predominant lesion formed following oxidative damage of DNA by reactive oxygen species, is processed differently by replicative and bypass polymerases. Our kinetic primer extension studies demonstrate that the bypass polymerase Dpo4 preferentially inserts C opposite oxoG, and also preferentially extends from the oxoG*C base pair, thus achieving error-free bypass of this lesion. We have determined the crystal structures of preinsertion binary, insertion ternary, and postinsertion binary complexes of oxoG-modified template-primer DNA and Dpo4. These structures provide insights into the translocation mechanics of the bypass polymerase during a complete cycle of nucleotide incorporation. Specifically, during noncovalent dCTP insertion opposite oxoG (or G), the little-finger domain-DNA phosphate contacts translocate by one nucleotide step, while the thumb domain-DNA phosphate contacts remain fixed. By contrast, during the nucleotidyl transfer reaction that covalently incorporates C opposite oxoG, the thumb-domain-phosphate contacts are translocated by one nucleotide step, while the little-finger contacts with phosphate groups remain fixed. These stepwise conformational transitions accompanying nucleoside triphosphate binding and covalent nucleobase incorporation during a full replication cycle of Dpo4-catalyzed bypass of the oxoG lesion are distinct from the translocation events in replicative polymerases.

The drug Premarin is the most widely used formula for hormone replacement therapy. However, long-term exposure to estrogens from the Premarin drug increases the risk of breast cancer. Equilin and equilenin, major components of Premarin, are predominantly metabolized to 4-hydroxyequilenin (4-OHEN). The quinoids produced by 4-OHEN oxidation react with dG, dA, and dC to form unusual stable cyclic bulky adducts, with four stereoisomers identified for each base adduct. The 4-OHEN-dC adducts are most predominant. They are mutagenic in vitro and have been found in human tumor tissue. We have carried out molecular modeling and molecular dynamics simulations to investigate structures and thermodynamics of the four 4-OHEN-dC stereoisomeric adducts in DNA duplexes. Our results show that the structure of each stereoisomer adduct in duplex DNA is specifically governed by its unique stereochemistry. The bulky adducts, with an obstructed Watson-Crick edge and an equilenin ring system near perpendicular to the damaged cytosine, are located in the B-DNA major or minor groove, with the modified cytosine in the syn or anti conformation, respectively. The DNA duplex structures are distorted, in terms of Watson-Crick pairing at and near the lesion, stacking interactions, and groove dimensions. Stereochemistry determines the orientation of the equilenin rings with respect to the 5'- to 3'-direction of the modified strand, as well as the positioning of the equilenin moiety's methyl and hydroxyl groups for each stereoisomer. The unusual structures and the stereochemical effects underlie their biological processing as miscoding DNA lesions whose mutagenic properties may contribute to breast cancer.

Equilenin, an important component of a widely prescribed hormone replacement formulation for postmenopausal women, is metabolized by mammalian P450 enzymes to the catechol 4-hydroxyequilenin (4-OHEN). The oxidized o-quinone derivative of 4-OHEN is known to form cyclic covalent adducts with DNA [Bolton, J. (1998) Chem. Res. Toxicol. 11, 1113] in vitro and in vivo. The characteristics of 4-OHEN-DNA adduct formation were investigated with the oligonucleotides 5'-d(CCATCGCTACC) (I), its complementary strand 5'-d(GGTAGCGATGG) (II), one rich in C and the other in G, and the duplexes I.II. The identities of the modified bases were elucidated in terms of four stereoisomeric 4-OHEN-2'-deoxynucleoside standards described earlier [Shen et al. (2001) Chem. Res. Toxicol. 11, 94; Embrechts et al. J. Mass Spectrom. 36, 317). The reactions of 4-OHEN with C are favored overwhelmingly in both single-stranded I and II with no guanine adducts observed in either case, and only minor proportions of A adducts were detected in sequence II. However, guanine adducts are observed in oligonucleotides that contain only G and unreactive T residues. The relative levels of cyclic covalent adducts observed in single-stranded I, II, and duplex I.II are approximately 54:21:5, with only the end C groups in I modified in the I.II duplex. When 4-OHEN is reacted with calf thymus DNA, the reaction yield of cyclic adducts is more than approximately 10(3)-fold lower than in I. The cyclic 4-OHEN adducts lead to a pronounced thermal destabilization of duplexes I.II. Overall, cyclic adduct formation is markedly dependent on the sequence context and secondary structure of the DNA. The latter effect is attributed to the poor accessibilities of 4-OHEN to the reactive nucleotide Watson-Crick hydrogen-bonding interface in the interior of the duplex. In the single-stranded oligonucleotides I and II, the strikingly different selectivities of adduct formation are attributed to the formation of noncovalent preassociation complexes that favor reaction geometries with C, rather than with A or G. Finally, the levels of several typical biomarkers of oxidative DNA damage (including 8-oxo-2'-deoxyguanosine) are formed in I in aqueous solutions with a yield at least 10 times smaller than the yield of cyclic 4-OHEN-dC adducts under identical reaction conditions.

Oxidation of guanine or 8-oxo-7,8-dihydroguanine can produce spiroiminodihydantoin (Sp) R and S stereoisomers. Both in vitro and in vivo experiments have shown that the Sp stereoisomers are highly mutagenic, causing G --> C and G --> T transversion mutations. Therefore, they are of interest as potential endogenous cancer causing lesions. However, their structural properties in DNA duplexes remain to be elucidated. We have employed computational methods to study the Sp lesions in 11-mer DNA duplexes with A, C, G, and T partners. Molecular dynamics simulations have been carried out to obtain ensembles of structures, and the trajectories were employed to analyze the structures and compute free energies. The structural and thermodynamic analyses reveal that the Sp stereoisomers energetically favor positioning in the B-DNA major groove, with minor groove conformers also low energy in some cases, depending on the partner base. The R and S stereoisomers adopt opposite orientations with respect to the 5' to 3' direction of the modified strand. Both syn and anti glycosidic bond conformations are energetically feasible, with partner base and stereochemistry determining the preference. The lesions adversely impact base stacking and Watson-Crick hydrogen bonding interactions in the duplex, and cause groove widening. The chemical nature of the partner base determines specific hydrogen bonding and stacking properties of the damaged duplexes. The structural characteristics may relate to observed mutagenic properties of the Sp stereoisomers, including possible stereoisomer-dependent differences.

8-Nitro-2'-deoxyguanosine (8-NO(2)-dG) DNA adducts are induced by the reactive nitrogen species and may be associated with the development of cancer in inflammatory tissues. To explore the miscoding potential of 8-NO(2)-dG adduct, an oligodeoxynucleotide containing a single 8-NO(2)-dG adduct was prepared by photochemical synthesis and used as a template in primer extension reactions catalyzed by mammalian DNA polymerases (pol). Primer extension reactions catalyzed by pol alpha or beta were strongly retarded at the 8-NO(2)-dG lesion; a fraction of primers was extended past the lesion by incorporating preferentially dCMP, the correct base, opposite the lesion, accompanied by lesser amounts of dAMP and dGMP incorporation. In contrast, primer extension reactions catalyzed by pol eta or a truncated form of pol kappa (pol kappaDeltaC) readily extended past the 8-NO(2)-dG lesion. Pol eta and kappaDeltaC showed more broad miscoding spectra; direct incorporations of dCMP and dAMP were observed, along with lesser amounts of dGMP and dTMP incorporations and deletions. The miscoding frequencies induced by pol eta and kappaDeltaC were at least 8 times higher than that of pol alpha or beta. Miscoding frequency and specificity of 8-NO(2)-dG varied depending on the DNA polymerases used. These observations were supported by steady-state kinetic studies. 8-NO(2)-dG adduct may play an important role in initiating inflammation driven carcinogenesis.

Reactive oxygen species present in the cell generate DNA damage. One of the major oxidation products of guanine in DNA, 8-oxo-7,8-dihydroguanine, formed by loss of two electrons, is among the most extensively studied base lesions. The further removal of two electrons from this product can yield spiroiminodihydantoin (Sp) R and S stereoisomers. Both in vitro and in vivo experiments have shown that the Sp stereoisomers are highly mutagenic, causing G --> T and G --> C transversions. Hence, they are of interest as examples of endogenous DNA damage that may initiate cancer. To interpret the mutagenic properties of the Sp lesions, an understanding of their structural properties is needed. To elucidate these structural effects, we have carried out computational investigations at the level of the Sp-modified base and nucleoside. At the base level, quantum mechanical geometry optimization studies have revealed exact mirror image symmetry of the R and S stereoisomers, with a near-perpendicular geometry of the two rings. At the nucleoside level, an extensive survey of the potential energy surface by molecular mechanics calculations using AMBER has provided three-dimensional potential energy maps. These maps reveal that the range and flexibility of the glycosidic torsion angles are significantly more restricted in both stereoisomeric adducts than in unmodified 2'-deoxyguanosine. The structural and energetic results suggest that the unusual geometric, steric, and hydrogen bonding properties of these lesions underlie their mutagenicity. In addition, stereoisomer-specific differences indicate the possibility that their processing by cellular replication and repair enzymes may be differentially affected by their absolute configuration.

It is well known that CpG dinucleotide steps in DNA, which are highly methylated at the 5-position of cytosine (meC) in human tissues, exhibit a disproportionate number of mutations within certain codons of the p53 gene. There is ample published evidence indicating that the reactivity of guanine with anti-B[a]PDE (a metabolite of the environmental carcinogen benzo[a]pyrene) at CpG mutation hot spots is enhanced by the methylation of the cytosine residue flanking the target guanine residue on the 5'-side. In this work we demonstrate that such a methylation can also dramatically affect the conformational characteristics of an adduct derived from the reaction of one of the two enantiomers of anti-B[a]PDE with the exocyclic amino group of guanine ([BP]G adduct). A detailed NMR study indicates that the 10R (-)-trans-anti-[BP]G adduct undergoes a transition from a minor groove-binding alignment of the aromatic BP ring system in the unmethylated C-[BP]G sequence context, to an intercalative BP alignment with a concomitant displacement of the modified guanine residue into the minor groove in the methylated meC-[BP]G sequence context. By contrast, a minor groove-binding alignment was observed for the stereoisomeric 10S (+)-trans-anti-[BP]G adduct in both the C-[BP]G and meC-[BP]G sequence contexts. This remarkable conformational switch resulting from the presence of a single methyl group at the 5-position of the cytosine residue flanking the lesion on the 5'-side, is attributed to the hydrophobic effect of the methyl group that can stabilize intercalated adduct conformations in an adduct stereochemistry-dependent manner. Such conformational differences in methylated and unmethylated CpG sequences may be significant because of potential alterations in the cellular processing of the [BP]G adducts by DNA transcription, replication, and repair enzymes.

Molecular modeling and molecular dynamics simulations have been performed to elucidate feasible structures in the Y-family Dpo4 DNA polymerase for the 1S-(-)-trans-anti-B[c]Ph-N6-dA adduct, derived from the fjord region polycyclic aromatic hydrocarbon (PAH) benzo[c]phenanthrene. Three types of models were delineated as follows: an intercalation model, a model with the aromatic ring system in the polymerase major groove open pocket, and a -1 deletion major groove model. All four 2'-deoxyribonucleoside 5'-triphosphates (dNTPs) were considered in the first two cases, and a normal Watson-Crick partner positioned to have skipped the modified template was employed as the incoming dNTP in the -1 deletion case. The trajectories derived from the dynamics simulations were analyzed in detail to evaluate the extents of distortion for each system. Overall, our results suggest that the major groove model is the least distorted, followed by the -1 deletion model, while the intercalation model is perturbed the most. The syn-dGTP and syn-dATP mismatches opposite the lesion are well-accommodated in the major groove model, as is the normal Watson-Crick partner dTTP. The intercalation model appears most likely to impede the polymerase. More broadly, these models look reasonable for other PAH metabolite-derived adducts to adenine with similar 1S stereochemistry. Furthermore, these models suggest how error-prone translesion synthesis by Y-family polymerases might produce mutations that may play a role in the initiation of cancer.

One of the major biomarkers of oxidative stress and oxidative damage of cellular DNA is 8-oxo-7,8-dihydroguanine (8-oxoGua), which is more easily oxidized than guanine to diverse oxidative products. In this work, we have investigated further oxidative transformations of 8-oxoGua in single- and double-stranded oligonucleotides to the dehydroguanidinohydantoin, oxaluric acid, and diastereomeric spiroiminodihydantoin lesions. The relative distributions of these end products were explored by a combined kinetic laser spectroscopy and mass spectrometry approach and are shown to depend markedly on the presence of superoxide radical anions. The 8-oxaGua radicals were produced by one-electron oxidation of 8-oxoGua by 2-aminopurine radicals generated by the two-photon ionization of 2-aminopurine residues site specifically positioned in 5'-d(CC[2-aminopurine]TC[8-oxoGua]CTACC). The hydrated electrons also formed in the photoionization process were trapped by dissolved molecular oxygen thus producing superoxide. A combination reaction between the 8-oxoGua and superoxide radicals occurs with the rate constant of (1.3 +/- 0.2) x 10(8) m(-1) s(-1) and (1.0 +/- 0.5) x 10(8) m(-1) s(-1) in single- and double-stranded DNA, respectively. The major end products of this reaction are the dehydroguanidinohydantoin lesions that slowly hydrolyze to oxaluric acid residues. In the presence of Cu,Zn-superoxide dismutase, an enzyme that induces the rapid catalytic dismutation of superoxide to the less reactive H(2)O(2) and O(2), the yields of the dehydroguanidinohydantion lesions become negligible. Under these conditions, the 8-oxoGua radicals do not exhibit any observable reactivities with oxygen (k < 10(2) m(-1) s(-1)), decay on the time interval of several seconds, and the major reaction products are the spiroiminodihydantoin lesions. The possible biological implications of the 8-oxoGua oxidation are discussed.

The oxidation and nitration reactions in DNA associated with the combination of nitrogen dioxide radicals with 8-oxo-7,8-dihydroguanine (8-oxoGua) and guanine radicals were explored by kinetic laser spectroscopy and mass spectrometry methods. The oxidation/nitration processes were triggered by photoexcitation of 2-aminopurine (2AP) residues site-specifically positioned in the 2'-deoxyribooligonucleotide 5'-d(CC[2AP]TC[X]CTACC) sequences (X = 8-oxoGua or G), by intense 308 nm excimer laser pulses. The photoionization products, 2AP radicals, rapidly oxidize either 8-oxoGua or G residues positioned within the same oligonucleotide but separated by a TC dinucleotide step on the 3'-side of 2AP. The two-photon ionization of the 2AP residue also generates hydrated electrons that are trapped by nitrate anions thus forming nitrogen dioxide radicals. The combination of nitrogen dioxide radicals with the 8-oxoGua and G radicals occurs with similar rate constants (approximately 4.3 x 10(8) M(-1) s(-1)) in both single- and double-stranded DNA. In the case of 8-oxoGua, the major end-products of this bimolecular radical-radical addition are spiroiminodihydantoin lesions, the products of 8-oxoGua oxidation. Oxygen-18 isotope labeling experiments reveal that the O-atom in the spiroiminodihydantoin lesion originates from water molecules, not from nitrogen dioxide radicals. In contrast, combination of nitrogen dioxide and guanine neutral radicals generated under the same conditions results in the formation of the nitro products, 5-guanidino-4-nitroimidazole and 8-nitroguanine adducts. The mechanistic aspects of the oxidation/nitration processes and their biological implications are discussed.

Of the carcinogens to which humans are most frequently exposed, the polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) is one of the most ubiquitous. BP is a byproduct of grilled foods and tobacco and fuel combustion and has long been linked to various human cancers, particularly lung and skin. BP is metabolized to diol epoxides that covalently modify DNA bases to form bulky adducts that block DNA synthesis by replicative or high fidelity DNA polymerases. Here we present the structure of a high fidelity polymerase from a thermostable strain of Bacillus stearothermophilus (Bacillus fragment) bound to the most common BP-derived N2-guanine adduct base-paired with cytosine. The BP adduct adopts a conformation that places the polycyclic BP moiety in the nascent DNA minor groove and is the first structure of a minor groove adduct bound to a polymerase. Orientation of the BP moiety into the nascent DNA minor groove results in extensive disruption to the interactions between the adducted DNA duplex and the polymerase. The disruptions revealed by the structure of Bacillus fragment bound to a BP adduct provide a molecular basis for rationalizing the potent blocking effect on replication exerted by BP adducts.

DNA methylation is an important cellular mechanism for controlling gene expression. Whereas the mutagenic properties of many DNA adducts, e.g., those arising from polycyclic aromatic hydrocarbons, have been widely studied, little is known about their influence on DNA methylation. We have constructed site-specifically modified 18-mer oligodeoxynucleotide duplexes containing a pair of stereoisomeric adducts derived from a benzo[a]pyrene-derived diol epoxide [(+)- and (-)-r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, or B[a]PDE] bound to the exocyclic amino group of guanine. The adducts, either (+)- or (-)-trans-anti-B[a]P-N(2)-dG (G*), positioned either at the 5'-side or the 3'-side deoxyguanosine residue in the recognition sequence of EcoRII restriction-modification enzymes (5'-...CCA/TGG...) were incorporated into 18-mer oligodeoxynucleotide duplexes. The effects of these lesions on complex formation and the catalytic activity of the EcoRII DNA methyltransferase (M.EcoRII) and EcoRII restriction endonuclease (R.EcoRII) were investigated. The M.EcoRII catalyzes the transfer of a methyl group to the C5 position of the 3'-side cytosine of each strand of the recognition sequence, whereas R.EcoRII catalyzes cleavage of both strands. The binding of R.EcoRII to the oligodeoxynucleotide duplexes and the catalytic cleavage were completely abolished when G was positioned at the 3'-side dG position (5'-...CCTGG*...). When G* was at the 5'-side dG position, binding was moderately diminished, but cleavage was completely blocked. In the case of M.EcoRII, binding is diminished by factors of 5-30 but the catalytic activity was either abolished or reduced 4-80-fold when the adducts were located at either position. Somewhat smaller effects were observed with hemimethylated oligodeoxynucleotide duplexes. These findings suggest that epigenetic effects, in addition to genotoxic effects, need to be considered in chemical carcinogenesis initiated by B[a]PDE, since the inhibition of methylation may allow the expression of genes that promote tumor development.

Human RPA (replication protein A), a single-stranded DNA-binding protein, is required for many cellular pathways including DNA repair, recombination and replication. However, the role of RPA in nucleotide excision repair remains elusive. In the present study, we have systematically examined the binding of RPA to a battery of well-defined ssDNA (single-stranded DNA) substrates using fluorescence spectroscopy. These substrates contain adducts of (6-4) photoproducts, N-acetyl-2-aminofluorene-, 1-aminopyrene-, BPDE (benzo[a]pyrene diol epoxide)- and fluorescein that are different in many aspects such as molecular structure and size, DNA disruption mode (e.g. base stacking or non-stacking), as well as chemical properties. Our results showed that RPA has a lower binding affinity for damaged ssDNA than for non-damaged ssDNA and that the affinity of RPA for damaged ssDNA depends on the type of adduct. Interestingly, the bulkier lesions have a greater effect. With a fluorescent base-stacking bulky adduct, (+)-cis-anti-BPDE-dG, we demonstrated that, on binding of RPA, the fluorescence of BPDE-ssDNA was significantly enhanced by up to 8-9-fold. This indicated that the stacking between the BPDE adduct and its neighbouring ssDNA bases had been disrupted and there was a lack of substantial direct contacts between the protein residues and the lesion itself. For RPA interaction with short damaged ssDNA, we propose that, on RPA binding, the modified base of ssDNA is looped out from the surface of the protein, permitting proper contacts of RPA with the remaining unmodified bases.

Remarkably different conformations can result when DNA binds with stereoisomeric compounds containing differing absolute configurations of substituents about chiral carbon atoms. Furthermore, the biochemical functions of covalent adducts with DNA are strongly affected by the stereochemistry of the ligands. Such stereochemical effects are manifested by DNA covalent adducts derived from metabolites of the non-planar fjord region environmental chemical carcinogen benzo[c]phenanthrene. To analyze these phenomena, an extensive conformational investigation for R and S stereoisomeric adducts to deoxyadenosine, derived from trans addition of enantiomeric anti diol epoxide metabolites of benzo[c]phenanthrene, has been carried out. We have surveyed the potential energy surface of the two adducts by varying systematically at 5 degree intervals in combination, the three important torsion angles that govern conformational flexibility of the carcinogen bulk with respect to the linked nucleoside. We carried out a grid search by creating 373, 248 structures for each isomer, and evaluated their molecular mechanical energies. This has permitted us to map the potential energy surface of each adduct in these three variables, and to delineate their low energy regions. The maps have a symmetric relationship which stems from the near mirror-image stereochemistry in the R and S isomers. This produces near mirror-image low energy structures in the nucleoside adducts. The limited sets of stereoisomer-dependent conformational domains delineated are determined by steric effects. Moreover, these features have been experimentally demonstrated to play governing structural roles in such carcinogen-damaged DNA duplexes: opposite orientations in the stereoisomer pairs computed for the nucleosides are observed by high-resolution NMR in the similarly modified DNA double helices, and are likely to play important roles in their interactions with enzymes involved in DNA transactions, and hence their biological activities.

Y-family DNA polymerases lack some of the mechanisms that replicative DNA polymerases employ to ensure fidelity, resulting in higher error rates during replication of undamaged DNA templates and the ability to bypass certain aberrant bases, such as those produced by exposure to carcinogens, including benzo[a]pyrene (BP). A tumorigenic metabolite of BP, (+)-anti-benzo-[a]pyrene diol epoxide, attacks DNA to form the major 10S (+)-trans-anti-[BP]-N(2)-dG adduct, which has been shown to be mutagenic in a number of prokaryotic and eukaryotic systems. The 10S (+)-trans-anti-[BP]-N(2)-dG adduct can cause all three base substitution mutations, and the SOS response in Escherichia coli increases bypass of bulky adducts, suggesting that Y-family DNA polymerases are involved in the bypass of such lesions. Dpo4 belongs to the DinB branch of the Y-family, which also includes E. coli pol IV and eukaryotic pol kappa. We carried out primer extension assays in conjunction with molecular modeling and molecular dynamics studies in order to elucidate the structure-function relationship involved in nucleotide incorporation opposite the bulky 10S (+)-trans-anti-[BP]-N(2)-dG adduct by Dpo4. Dpo4 is able to bypass the 10S (+)-trans-anti-[BP]-N(2)-dG adduct, albeit to a lesser extent than unmodified guanine, and the V(max) values for insertion of all four nucleotides opposite the adduct by Dpo4 are similar. Computational studies suggest that 10S (+)-trans-anti-[BP]-N(2)-dG can be accommodated in the active site of Dpo4 in either the anti or syn conformation due to the limited protein-DNA contacts and the open nature of both the minor and major groove sides of the nascent base pair, which can contribute to the promiscuous nucleotide incorporation opposite this lesion.

In living tissues under inflammatory conditions, superoxide radicals (O(2)*)) are generated and are known to cause oxidative DNA damage. However, the mechanisms of action are poorly understood. It is shown here that the combination of O(2)* with guanine neutral radicals, G(-H)* in single- or double-stranded oligodeoxyribonucleotides (rate constant of 4.7 +/- 1.0 x 10(8) m(-1) s(-1) in both cases), culminates in the formation of oxidatively modified guanine bases (major product, imidazolone; minor product, 8-oxo-7,8-dihydroguanine). The G(-H)* and O(2)* radicals were generated by intense 308 nm excimer laser pulses resulting in the one-electron oxidation and deprotonation of guanine in the 5'-d(CC[2AP]-TCGCTACC) strands and the trapping of the ejected electrons by molecular oxygen (Shafirovich, V., Dourandin, A., Huang, W., Luneva, N. P., and Geacintov, N. E. (2000) Phys. Chem. Chem. Phys. 2, 4399-4408). The addition of Cu,Zn-superoxide dismutase, known to react rapidly with superoxide, dramatically enhances the life-times of guanine radicals from 4 to 7 ms to 0.2-0.6 s in the presence of 5 microm superoxide dismutase. Oxygen-18 isotope labeling experiments reveal two pathways of 8-oxo-7,8-dihydroguanine formation including either addition of O(2)* to the C-8 position of G(-H)* (in the presence of oxygen), or the hydration of G(-H)* (in the absence of oxygen). The formation of the guanine lesions via combination of guanine and superoxide radicals is greatly reduced in the presence of typical antioxidants such as trolox and catechol that rapidly regenerate guanine by the reductive "repair" of G(-H)* radicals. The mechanistic aspects of the radical reactions that either regenerate undamaged guanine in DNA or lead to oxidatively modified guanine bases are discussed.

Fidelity of DNA polymerases is predominantly governed by an induced fit mechanism in which the incoming dNTP in the ternary complex fits tightly into a binding pocket whose geometry is determined by the nature of the templating base. However, modification of the template with a bulky carcinogen may alter the dNTP binding pocket and thereby the polymerase incorporation fidelity. High fidelity DNA polymerases, such as bacteriophage T7 DNA polymerase, are predominantly blocked by bulky chemical lesions on the template strand during DNA replication. However, some mutagenic bypass can occur, which may lead to carcinogenesis. Experimental studies have shown that a DNA covalent adduct derived from (+)-anti-BPDE [(+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene], a carcinogenic metabolite of benzo[a]pyrene (BP), primarily blocks Sequenase 2.0, an exo(-) T7 DNA polymerase; however, a mismatched dATP can be preferentially inserted opposite the damaged adenine templating base within the active site of the polymerase [Chary, P., and Lloyd, R. S. (1995) Nucleic Acids Res. 23, 1398-1405]. The goal of this work is to elucidate structural features that contribute to DNA polymerase incorporation fidelity in the presence of this bulky covalent adduct and to interpret the experimental findings on a molecular level. We have carried out molecular modeling and molecular dynamics simulations with AMBER 6.0, investigating a T7 DNA polymerase primer-template closed ternary complex containing this 10S (+)-trans-anti-[BP]-N(6)-dA adduct in the templating position within the polymerase active site. All four incoming dNTPs were studied. The simulations show that the BP ring system fits well into an open pocket on the major groove side of the modified template adenine with anti glycosidic bond conformation, without disturbing critical polymerase-DNA interactions. However, steric hindrance between the BP ring system and the primer-template DNA causes displacement of the modified template adenine, so that the dNTP base binding pocket is enlarged. This alteration can explain the experimentally observed preference for incorporation of dATP opposite this lesion. These studies also rationalize the observed lower probabilities of incorporation of the other three nucleotides. Our results suggest that the differences in incorporation of dGTP, dCTP, and dTTP are due to the effects of imperfect geometric complementarity. Thus, the simulations suggest that altered DNA polymerase incorporation fidelity can result from adduct-induced changes in the dNTP base binding pocket geometry. Furthermore, plausible structural explanations for the observed effects of [BP]-N(6)-dA adduct stereochemistry on the observed stalling patterns are proposed.

The DNA repair protein UvrB plays an indispensable role in the stepwise and sequential damage recognition of nucleotide excision repair in Escherichia coli. Our previous studies suggested that UvrB is responsible for the chemical damage recognition only upon a strand opening mediated by UvrA. Difficulties were encountered in studying the direct interaction of UvrB with adducts due to the presence of UvrA. We report herein that a single point mutation of Y95W in which a tyrosine is replaced by a tryptophan results in an UvrB mutant that is capable of efficiently binding to structure-specific DNA adducts even in the absence of UvrA. This mutant is fully functional in the UvrABC incisions. The dissociation constant for the mutant-DNA adduct interaction was less than 100 nM at physiological temperatures as determined by fluorescence spectroscopy. In contrast, similar substitutions at other residues in the beta-hairpin with tryptophan or phenylalanine do not confer UvrB such binding ability. Homology modeling of the structure of E. coli UvrB shows that the aromatic ring of residue Y95 and only Y95 directly points into the DNA binding cleft. We have also examined UvrB recognition of both "normal" bulky BPDE-DNA and protein-cross-linked DNA (DPC) adducts and the roles of aromatic residues of the beta-hairpin in the recognition of these lesions. A mutation of Y92W resulted in an obvious decrease in the efficiency of UvrABC incisions of normal adducts, while the incision of the DPC adduct is dramatically increased. Our results suggest that Y92 may function differently with these two types of adducts, while the Y95 residue plays an unique role in stabilizing the interaction of UvrB with DNA damage, most likely by a hydrophobic stacking.

Carbonate radical anions are potentially important oxidants of nucleic acids in physiological environments. However, the mechanisms of action are poorly understood, and the end products of oxidation of DNA by carbonate radicals have not been characterized. These oxidation pathways were explored in this work, starting from the laser pulse-induced generation of the primary radical species to the identification of the stable oxidative modifications (lesions). The cascade of events was initiated by utilizing 308 nm XeCl excimer laser pulses to generate carbonate radical anions on submicrosecond time scales. This laser flash photolysis method involved the photodissociation of persulfate to sulfate radical anions and the one electron oxidation of bicarbonate anions by the sulfate radicals to yield the carbonate radical anions. The latter were monitored by their characteristic transient absorption band at 600 nm. The rate constants of reactions of carbonate radicals with oligonucleotides increase in the ascending order: 5'-d(CCATCCTACC) [(5.7 +/- 0.6) x 10(6) M(-)(1) s(-)(1)] < 5'-d(TATAACGTTATA), self-complementary duplex [(1.4 +/- 0.2) x 10(7) M(-)(1) s(-)(1)] < 5'-d(CCATCGCTACC [(2.4 +/- 0.3) x 10(7) M(-)(1) s(-)(1)] < 5'-d(CCATC[8-oxo-G]CTACC) [(3.2 +/- 0.4) x 10(8) M(-)(1) s(-)(1)], where 8-oxo-G is 8-oxo-7,8-dihydroguanine, the product of a two electron oxidation of guanine. This remarkable enhancement of the rate constants is correlated with the presence of either G or 8-oxo-G bases in the oligonucleotides. The rate constant for the oxidation of G in a single-stranded oligonuclotide is faster by a factor of approximately 2 than in the double-stranded form. The site selective oxidation of G and 8-oxo-G residues by carbonate radicals results in the formation of unique end products, the diastereomeric spiroiminodihydantoin (Sp) lesions, the products of a four electron oxidation of guanine. These lesions, formed in high yields (40-60%), were isolated by reversed phase HPLC and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. These assignments were supported by the characteristic circular dichroism spectra of opposite signs of the two lesions. The oxidation of guanine to Sp diastereomers occurs, at least in part, via the formation of 8-oxo-G lesions as intermediates. The Sp lesions can be considered as the terminal products of the oxidation of G and 8-oxo-G in DNA by carbonate radical anions. The mechanistic aspects and biological implications of these site selective reactions in DNA initiated by carbonate radicals are discussed.

DNA damage recognition of nucleotide excision repair (NER) in Escherichia coli is achieved by at least two steps. In the first step, a helical distortion is recognized, which leads to a strand opening at the lesion site. The second step involves the recognition of the type of chemical modification in the single-stranded region of DNA during the processing of the lesions by UvrABC. In the current work, by comparing the efficiencies of UvrABC incision of several types of different DNA adducts, we show that the size and position of the strand opening are dependent on the type of DNA adducts. Optimal incision efficiency for the C8-guanine adducts of 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) was observed in a bubble of three mismatched nucleotides, whereas the same for C8-guanine adduct of 1-nitropyrene and N(2)-guanine adducts of benzo[a]pyrene diol epoxide (BPDE) was noted in a bubble of six mismatched nucleotides. This suggests that the size of the aromatic ring system of the adduct might influence the extent and number of bases associated with the opened strand region catalyzed by UvrABC. We also showed that the incision efficiency of the AF or AAF adduct was affected by the neighboring DNA sequence context, which, in turn, was the result of differential binding of UvrA to the substrates. The sequence context effect on both incision and binding disappeared when a bubble structure of three bases was introduced at the adduct site. We therefore propose that these effects relate to the initial step of damage recognition of DNA structural distortion. The structure-function relationships in the recognition of the DNA lesions, based on our results, have been discussed.

Environmental polycyclic aromatic hydrocarbons (PAHs) are metabolically activated to diol epoxides that can react with DNA, resulting in covalent modifications to the bases. The (+)- and (-)-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydro-benzo[c]phenanthrene (anti-BPhDE) isomers are diol epoxide metabolites of the PAH benzo[c]phenanthrene (BPh). These enantiomers readily react with DNA at the N6 position of adenine, forming bulky (+)-1R- or (-)-1S-trans-anti-[BPh]-N6-dA adducts. Transcription-coupled nucleotide excision repair clears such bulky adducts from cellular DNA, presumably in response to RNA polymerase transcription complexes that stall at the bulky lesions. Little is known about the effects of [BPh]-N6-dA lesions on RNA polymerase II, hence, the behavior of human RNA polymerase II was examined at these adducts. A site-specific, stereochemically pure [BPh]-N6-dA adduct was positioned on the transcribed or non-transcribed strand of a DNA template with a suitable promoter for RNA polymerase II located upstream from the lesion. Transcription reactions were then carried out with HeLa nuclear extract. Each [BPh]-dA isomer strongly impeded human RNA polymerase II progression when it was located on the transcribed strand; however, a small but significant degree of lesion bypass occurred, and the extent of polymerase blockage and bypass was dependent on the stereochemistry of the adduct. Molecular modeling of the lesions supports the idea that each adduct can exist in two orientations within the polymerase active site, one that permits nucleotide incorporation and another that blocks the RNA polymerase nucleotide entry channel, thus preventing base incorporation and causing the polymerase to stall or arrest.

Benzo[a]pyrene is a potent environmental carcinogen, which can be metabolized in cells to the DNA damaging agent anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (anti-BPDE). We hypothesize that mutations induced by BPDE DNA adducts are mainly generated through an error-prone translesion synthesis that requires a specialized DNA polymerase (Pol). Using an in vivo mutagenesis assay in the yeast model system, we have examined the potential roles of Pol(zeta) and Pol(eta) in (+/-)-anti-BPDE-induced mutagenesis. In cells proficient in mutagenesis, (+/-)-anti-BPDE induced 85% base substitutions with predominant G --> C followed by G --> T transversions, 9% deletions of 1-3 nucleotides, and 6% insertions of 1-3 nucleotides. In rad30 mutant cells lacking Pol(eta), (+/-)-anti-BPDE-induced mutagenesis was reduced and accompanied by a moderate decrease in base substitutions and more significant decrease in deletions and insertions of 1-3 nucleotides. In rev3 mutant cells lacking Pol(zeta), (+/-)-anti-BPDE-induced mutagenesis was mostly abolished, leading to a great decrease in both base substitutions and deletions/insertions of 1-3 nucleotides. In contrast, large deletions/insertions were significantly increased in cells lacking Pol(zeta). Consistent with the in vivo results, purified yeast Pol(zeta) performed limited translesion synthesis opposite (+)- and (-)-trans-anti-BPDE-N(2)-dG DNA adducts with predominant G incorporation opposite the lesion. These results show that (+/-)-anti-BPDE-induced mutagenesis in yeast requires Pol(zeta) and partially involves Pol(eta) and suggest that Pol(zeta) directly participates in nucleotide insertions opposite the lesion, while Pol(eta) significantly contributes to deletions and insertions of 1-3 nucleotides.

A simple photochemical approach is described for synthesizing site specific, stable 5-guanidino-4-nitroimidazole (NIm) adducts in single- and double-stranded oligodeoxynucleotides containing single and multiple guanine residues. The DNA sequences employed, 5'-d(ACC CG(1)C G(2)TC CG(3)C G(4)CC) and 5'-d(ACC CG(1)C G(2)TC C), were a portion of exon 5 of the p53 tumor suppressor gene, including the codons 157 (G(2)) and 158 (G(3)) mutation hot spots in the former sequence with four Gs and the codon 157 (G(2)) mutation hot spot in the latter sequence with two Gs. The nitration of oligodeoxynucleotides was initiated by the selective photodissociation of persulfate anions to sulfate radicals induced by UV laser pulses (308 nm). In aqueous solutions, of bicarbonate and nitrite anions, the sulfate radicals generate carbonate anion radicals and nitrogen dioxide radicals by one electron oxidation of the respective anions. The guanine residue in the oligodeoxynucleotide is oxidized by the carbonate anion radical to form the neutral guanine radical. While the nitrogen dioxide radicals do not react with any of the intact DNA bases, they readily combine with the guanine radicals at either the C8 or the C5 positions. The C8 addition generates the well-known 8-nitroguanine (8-nitro-G) lesions, whereas the C5 attack produces unstable adducts, which rapidly decompose to NIm lesions. The maximum yields of the nitro products (NIm + 8-nitro-G) were typically in the range of 20-40%, depending on the number of guanine residues in the sequence. The ratio of the NIm to 8-nitro-G lesions gradually decreases from 3.4 in the model compound, 2',3',5'-tri-O-acetylguanosine, to 2.1-2.6 in the single-stranded oligodeoxynucleotides and to 0.8-1.1 in the duplexes. The adduct of the 5'-d(ACC CG(1)C G(2)TC C) oligodeoxynucleotide containing the NIm lesion in codon 157 (G(2)) was isolated in HPLC-pure form. The integrity of this adduct was established by a detailed analysis of exonuclease digestion ladders by matrix-assisted laser desorption ionization with time-of-flight detection MS techniques.

Exposure to estrogen through estrogen replacement therapy increases the risk of women developing cancer in hormone sensitive tissues. Premarin (Wyeth), which has been the most frequent choice for estrogen replacement therapy in the United States, contains the equine estrogens equilin and equilenin as major components. 4-Hydroxyequilenin (4-OHEN) is a phase I metabolite of both of these substances. This catechol estrogen autoxidizes to potent cytotoxic quinoids that can react with dG, dA, and dC to form unusual stereoisomeric cyclic adducts (Bolton, J. L., et al. (1998) Chem. Res. Toxicol. 11, 1113-1127). Like other bulky DNA adducts, these lesions may exhibit different susceptibilities to DNA repair and mutagenic potential, if not repaired in a structure-dependent manner. To ultimately gain insights into structure-function relationships, we computed conformations of stereoisomeric guanine, adenine, and cytosine base adducts using density functional theory. We find near mirror image conformations in stereoisomer adduct pairs for each modified base, suggesting opposite orientations with respect to the 5' --> 3' direction of the modified strand when the stereoisomer pairs are incorporated into duplex DNA. Such opposite orientations could cause stereoisomer pairs of lesions to respond differently to DNA replication and repair enzymes.

A pair of stereoisomeric covalent adducts to guanine in double-stranded DNA, derived from the reaction of mutagenic and tumorigenic metabolites of benzo[a]pyrene, have been well characterized structurally and thermodynamically. Both high-resolution NMR solution structures and an array of thermodynamic data are available for these 10S (+)- and 10R (-)-trans-anti -[BP]-N(2)-dG adducts in double-stranded deoxyoligonucleotides. The availability of experimentally well-characterized duplexes containing these two stereoisomeric guanine adducts provides an opportunity for evaluating the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method for computing thermodynamic properties from molecular dynamics ensembles. We have carried out 3-ns molecular dynamics simulations, using NMR solution structures as the starting models for the 10S (+)- and 10R (-)-trans-anti-dG adducts in a DNA duplex 11-mer using AMBER 6.0. We employed the MM-PBSA method to compute the free energies, enthalpies, and entropies of the two adducts. Our complete thermodynamic analysis agrees quite well with the full experimental thermodynamic characterization of these adducts, showing essentially equal stabilities of the two adducts. We also calculated the nuclear Overhauser effect (NOE) distances from the molecular dynamics trajectories, and compared them against the experimental NMR-derived NOE distances. Our results showed that the simulated structures are in good agreement with the NMR experimental NOE data. Furthermore, the molecular dynamics simulations provided new structural and biological insights. Specifically, the puzzling observation that the BP aromatic ring system in the 10S (+)-trans-anti-dG adduct is more exposed to the aqueous solvent than the 10R (-)-trans-anti-dG adduct, is rationalized in terms of the adduct structures. The structural and thermodynamic features of these stereoisomeric adducts are also discussed in relation to their reported low susceptibilities to nucleotide excision repair.